We have utilized molecular techniques to gain new insights into the behavior of hematopoietic stem and progenitor cells (HSPCs) in vivo. We have continued active development and utilization of lentiviral barcoding with high-diversity 31-35 base pair genetic barcodes utilized to label individual hematopoietic stem and progenitor cells to study in vivo hematopoiesis in the non-human primate model. Our collaborator Rong Lu first devised this very powerful approach and applied it to study murine hematopoiesis. We have now transplanted 15 macaques with barcoded autologous CD34+ cells, and have been able to track hematopoietic output from thousands of individual HSPCs over time (up to 4.5 years) and in multiple lineages in a quantitative and highly reproducible manner. We have already made a number of important and novel discoveries, including the lack of evidence for a common lymphoid progenitor producing T and B cells in primates, with no shared clonal derivation of B and T cells until late after transplant, and much earlier shared clonal derivation of myeloid and B cells. We have also for the first time discovered the unique lineage derivation and life history of the major fraction of natural killer (NK) cells. CD16+CD56- cytotoxic NK cells did not share barcodes with B, T or myeloid cells even 48 months post-transplant. In vitro and murine models have not previously been able to shed light on NK cell lineage relationships. We have continued to use barcoded cells from these macaques to further dissect in vivo NK cell ontogeny, and the process of ex vivo expansion of NK cells, highly relevant for adoptive cell therapy development, in collaboration with Dr. Rick Childs' group in NHLBI, Dr Miti Kaur's group at the Tulane Non-human Primate facility, and Dr Yenan Bryceson's group at the Karolinska Institut in Stockholm Sweden. We have mapped the clonally-unique barcodes to NK cells with an adaptive phenotype, linked in murine studies to NK memory, and in humans to putative NK adaptive responses to viruses such as CMV. Within NK cells with an adaptive phenotype, we find oligoclonal and massively expanded individual clones, with no contributions found in other lineages, and with waxing and waning patterns suggesting responses to specific environmental cues such as viral infection or reactivation. Our data provides the first direct demonstration of clonal NK responses, possibly explaining NK memory. We continue to search for the precursor to these cells, tracking dominant clones in phenotypically purified samples from blood, bone marrow, lymph nodes, liver, and vaginal and intestinal lymphoid aggregates. With profound in vivo NK depletion, the same clones arise again, without recruitment from highly polyclonal HSPC. We have extended our analysis of the geographic segregation of individual HSPCs long term in specific marrow sites, confirming our prior findings in mice using LEGO imaging techniques now in the macaque model utilizing barcoding. We can directly demonstrate in situ production of B cells, CD56+ NK cells and myeloid cells in localized marrow niches, and surprisingly, we now have strong evidence for in situ marrow production of T cells. We have also followed the output of thousands of individual HSPC clones in several animals for up to 4 years, and demonstrate marked clonal stability of output from long-term repopulating clones producing myeloid, B cell and T cell lineages, along with CD56brightCD16- NK cells. We have shown that myeloid vs lymphoid bias is common, and that this bias remains stable in individual clones over time. We have recently extended the barcoding model in a number of additional directions, including: 1) Comparison of the clonal behavior of young versus aged HSPC, with preliminary data from transplants of two aged macaques with barcoded cells demonstrating a very different kinetic and clonal pattern compared to young animals. 2) Analysis of novel methodologies for stem cell expansion, with quantitative and lineage analytics performed on expanded versus non-expanded cells in vivo. 3) Investigation of the clonal ontogeny of erythroid and platelet lineages. 4) Collaboration with Rahul Sajita at NYU to apply single cell RNAseq to barcoded populations in order to further define ontogeny as well as identify rare precursor cell populations. We now have set up the DropSeq single cell methodology at NHLBI in collaboration with Andre Larochelle, and have evidence that we can retrieve both the barcode and the transcriptome from single cells. This approach offers many investigative opportunities.